S.S. Keswani1, A. Degala1, N. Nanda1, P. Bommekal1, J. Tellez1, I. Huddle1, S. Sinhas1, V. Akondy1, H. Li1, L. Yu1, M. Guerra1, M. Wietecha3, P. Bollyky2, S. Balaji1, S. Keswani1 1Baylor College Of Medicine, Pediatric Surgery, Houston, TX, USA 2Standford University School of Medicine, Infectious Disease, Stanford, CA, USA 3University Of Illinois at Chicago, Dentistry, Chicago, IL, USA
Introduction:
We have reported the critical role of CD4+ T lymphocytes in dermal wound healing and fibrosis, demonstrating that Tr1 lymphocytes (CD4+, CD44+, CD25+, FOXP3-) can promote regenerative repair. However, how Tr1 cells directly affect dermal fibroblasts (Fb), the main cellular arbiters of extracellular matrix (ECM) remodeling and fibrosis as part of their regenerative mechanisms remains unknown. We hypothesize that Tr1 cells directly influence fibroblast ECM production and drive distinct Fb subpopulations to promote regenerative wound repair.
Methods:
In vitro CD4+ total lymphocytes (T naïve) and Tr1 cells were isolated from 10bit (C57) mice, flow-sorted, and activated in culture. Conditioned media (CM) from days 6-9 was transferred to C57-murine dermal fibroblasts (mDFb) for 24hrs and assessed for gene expression of fibrosis-associated markers, Col1a1, Col3a1, TGFb1, and CTGF. Human dermal fibroblasts (hDFb) were isolated from patients with high or low scarring, treated with Tr1 CM, and assessed for similar endpoints to validate the effects of Tr1. To assess Tr1 effects on fibroblast populations, SCID mice were adoptively transferred with 1M T naive or Tr1 cells and 6mm wounds were created. Wounds were assessed at D7 and D28 by 10x Visium RNA sequencing and analyzed using R and Seurat workflows and histology. n=3/group, Data presented as mean ± SD, p values by t-test.
Results:
Tr1CM-treated mDFb showed a 3.8-fold higher Col3a1/Col1a1 ratio, an anti-fibrotic pattern, and decreased expression of pro-fibrotic TGFb1 and CTGF compared to T naïve treated Fb. Similarly, Tr1 CM-treatment of high scarring hDFb reduced the pro-fibrotic gene expression of all the fibrotic genes tested. In vivo, adoptive transfer of T lymphocyte subsets uniquely influenced the geospatial distribution of cell populations within the wound. 10x Visium single-cell data showed distinct patterns of gene enrichments in D7 SCID wounds treated with different lymphocyte subsets, with Tr1 wounds showing decreased inflammatory signature and more smooth muscle and adipose tissue (Fig. A). Also, module scoring of top up-regulated genes in high vs. low scar-forming hDFb compared to patterns in murine wounds showed less association in Tr1-treated compared to control lymphocyte-treated wounds (Fig. B), suggesting Tr1 reduces fibrotic fibroblast gene signature.
Conclusion:
Tr1 cells impact fibroblasts by downregulating profibrotic genes, with unique effects on phenotype and geospatial cellular distribution within wounds. These data provide a novel mechanistic basis for the regenerative effects of Tr1 cells, emphasize the role of driving spatial fibroblast heterogeneity in wound repair, and support the development of Tr1s as a novel antifibrotic therapy.